Ex-vivo and in vivo factor XII gene therapy for hemophilia A and B

Abstract

Bypass activity for hemophilia A and B can be generated by natural or recombinant Factor VIIa. Factor XIIa when implanted into a guinea pig or monkey also facilitates the conversion of endogenous factor VII to VIIa, thereby providing bypass activity. Additionally, certain modified versions of Factor XII are known to be intrinsically active, with properties like Factor XIIa. Administration of unencapsulated Factor XIIa to a guinea pig causes a transient increase in plasma bypass activity. A continuous source of Factor XIIa, as provided by a gene therapy, is therapeutic for both Hemophilia A and B. There are three ways to provide for gene therapy. In each case, the gene for Factor XII (or Factor XIIa) can be introduced into the cell by the usual means, including, but not limited to, as naked DNA, as a DNA/lipid mixture, or as part of a viral vector system. In one manifestation, cells can be transfected with full length or modified versions of Factor XII, ex-vivo, and allowed to continuously express versions of recombinant Factor XII from unencapsulated recombinant cells implanted in the body of the patient. A second mechanism would require encapsulating the cells within the body. As a third mechanism of introducing Factor XIIa into the patient, full length or modified versions of the gene for human Factor XII can be directly administered in vivo. The advantage is provision of a universal gene therapy for hemophilia A and B, rather than separate gene therapies involving either Factor VIII (Hemophilia A) or Factor IX (Hemophilia B).

What is claimed:  
     
         1 . A factor XIIa-coated solid support comprising a recombinant factor XII polypeptide:  
     
     
         2 . The Factor XIIa-coated solid support of  claim 1 , wherein the polypeptide is a truncated polypeptide.  
     
     
         3 . The Factor XIIa-coated solid support of  claim 2 , wherein the truncated polypeptide comprises a sequence of amino acid residues 1-215 of SEQ ID No. 2.  
     
     
         4 . The Factor XIIa-coated solid support of  claim 2 , wherein the truncated polypeptide comprises a sequence of amino acid residues of about 1-196 of SEQ ID No. 2.  
     
     
         5 . The Factor XIIa-coated solid support of  claim 1 , wherein the solid support is a bead.  
     
     
         6 . A Factor XII-expressing cell comprising an expression vector construct containing a gene encoding a Factor XII polypeptide.  
     
     
         7 . The Factor XII-expressing cell of  claim 6 , wherein the polypeptide is a truncated polypeptide.  
     
     
         8 . The Factor XII-expressing cell of  claim 7 , wherein the truncated polypeptide comprises a sequence of amino acid residues 1-215 of SEQ ID No. 2.  
     
     
         9 . The Factor XII-expressing cell of  claim 7 , wherein the truncated polypeptide comprises a sequence of amino acid residues of about 1-196 of SEQ ID No. 2.  
     
     
         10 . The Factor XII-expressing cell of  claim 6 , wherein the cell comprises a blood cell, a hepatocyte, a fibroblast, and an endothelial cell.  
     
     
         11 . A method of treating a subject with a coagulation protein comprising providing a gene prepared as a naked DNA vector and administering the vector to the subject in a therapeutically effective amount, wherein the gene encodes a Factor XII polypeptide.  
     
     
         12 . A method of treating a subject with a coagulation protein comprising the steps of 
 a) providing a gene for a coagulation protein prepared as an expression vector construct;    b) transfecting host cells with the construct,    c) adding cells of step b) expressing the coagulation protein to a porous chamber; and    d) implanting the chamber into the subject, wherein the chamber is implanted in fluid communication with the bloodstream and inactive Factor VII in the bloodstream passing through the chamber becomes activated upon contact with the coagulation protein.    
     
     
         13 . The method of  claim 12 , wherein the coagulation protein is a Factor XII polypeptide.  
     
     
         14 . The method of  claim 13 , wherein the polypeptide is a truncated polypeptide.  
     
     
         15 . The method of  claim 14 , wherein the truncated polypeptide comprises a sequence of amino acid residues of about 1-196 of SEQ ID No. 2.  
     
     
         16 . The method of  claim 12 , wherein the chamber comprises a permeable membrane having one or more walls, defining a hollow chamber therewithin, a plurality of holes extending through the membrane and permitting fluid to enter and exit the membrane, each of the holes being sized so that it is just large enough to permit Factor VII to enter the chamber of the membrane, and activated Factor VIIa to exit the chamber, and an effective amount of the coagulation protein or a source of the coagulation protein being expressed by the cells.  
     
     
         17 . The method of  claim 16 , wherein the permeable membrane is a dialysis membrane formatted as a geometric filter comprising polymethacrylate or polycarbonate, or a statistical hole filter comprising cellulose acetate.  
     
     
         18 . The method of  claim 12 , wherein the chamber comprises a region for affixing the chamber to an abdominal wall and for removing or adding materials such as a diaphragm for penetration of a syringe.  
     
     
         19 . The method of  claim 12 , wherein the expression vector construct of a) is a naked DNA.  
     
     
         20 . The method of  claim 12 , wherein the expression vector construct of a) is encapsulated by a liposome.  
     
     
         21 . The method of  claim 12 , wherein the expression vector construct of a) is a viral vector.  
     
     
         22 . A method of treatment with a coagulation protein comprising administering to a subject, a therapeutically effective amount of a recombinant Factor XII polypeptide.